Macrophage migration inhibitory factor (MIF) is a cytokine secreted by activated lymphocytes and macrophages that critically regulates inflammation. As a multi-functional protein, MIF is thought to act as a major regulator of inflammation and a central upstream mediator of innate immune responses (Calandra, T., et al., Nat. Rev. Immunol. 2003, 3, 791-800), and play an important role in downstream signaling events via its binding with its known receptors CD74, CXCR2 and CXCR4 (Leng, L., et al., J. Exp. Med. 2003, 197(11), 1467-76; Gore, Y., et al., J. Biol. Chem. 2008, 283, 2784-92; Bernhagen, J., et al., Nat. Med. 2007, 13, 587-596; Cho, Y., et al., Proc. Natl. Acad. Sci. USA 2010, 107, 11313-8; McLean, L. R., et al., Bioorg. Med. Chem. Lett. 2010, 20, 1821-4; Weber, C., et al., Proc. Natl. Acad. Sci. USA 2008, 105, 16278-83).
Among cytokines, MIF is unique in that it functions as an enzyme exhibiting tautomerase catalytic activity. Structure analysis demonstrates that MIF exists as a homotrimer with the active site for the tautomerase activity located between two adjacent monomers of the homotrimer (Lubetsky, J. B., et al., Biochemistry 1999, 38, 7346-54). MIF catalyzes the tautomerization of D-dopachrome, phenylpyruvate, and certain catecholamines. (S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF tautomerase activity, binds to the same position of the active site as does p-hydroxyphenylpyruvic acid (a substrate of MIF), inhibits the binding of MIF to the ectodomain of its receptor CD74, and antagonizes biological activities of MIF (Leng, L., et al., J. Immunol. 2011, 186, 527-538; Lubetsky, J. B., et al., J. Biol. Chem. 2002, 277, 24976-82). Interestingly, N-acetyl-p-benzoquinone imine (NAPQI), a metabolite of acetaminophen, forms a covalent complex with MIF at its active site to irreversibly inhibit MIF tautomerase activity and biological effects of MIF (Senter, P. D., et al., Proc. Natl. Acad. Sci. USA 2002, 99, 144-9). Additionally, other small molecules have been shown to form covalent complexes with MIF and irreversibly inhibit MIF's tautomerase activity (Winner, M., et al., Cancer Res. 2008, 68, 7253-7257; Cross, J. V., et al., Biochem. J. 2009, 423, 315-321). Because small molecule inhibitors of MIF tautomerase have been reported to antagonize the biological function of MIF, inhibition of MIF's tautomerase activity has been used to screen for drug candidates for treating inflammatory and neoplastic disease (Garai, J., et al., Curr. Med. Chem. 2009, 16, 1091-1114; Xu, L., et al., Drug Discovery Today 2013, 18, 592-600). Recent studies of the P1G mutation in a mouse breast cancer model suggest that MIF tautomerase activity is an important determinant of tumor growth and metastasis (Simpson, K. D., et al., J. Immunol. 2012, 189, 5533-5540). Moreover, small molecule reversible MIF tautomerase inhibitors, by their direct and/or indirect enhancement of CD8+ T-cell immune responses, could surprisingly fill an unmet need for safer and more efficacious agents to treat cancer and other proliferative diseases, prevent metastasis, and also provide safer and more efficacious agents for treating and preventing infections by viruses, plasmodia, fungi, mycobacteria, helminths and other microbes whose clearance is enhanced by a CD8+ T-cell immune response. Additionally, the ability of an anti-MIF antibody or a small molecule MIF inhibitor to reduce airway hyper-responsiveness and airway remodeling in mouse models of asthma suggests that MIF tautomerase inhibitors could also have utility in treating asthma (see e.g. Amano, T., et al., Inflam. Res. 2007, 56, 24-31; Chen, P.-F., et al., Mol. Med., 2010, 16, 400-408).